One of the various approaches to immunising against N. meningitidis infection is to use outer membrane vesicles (OMVs). An efficacious OMV vaccine against serogroup B has been produced by the Norwegian National Institute of Public Health [e.g. ref. 1] but, although this vaccine is safe and prevents NmB disease, its efficacy is limited to the strain used to make the vaccine.
The ‘RIVM’ vaccine is based on vesicles containing six different PorA subtypes and has been shown to be immunogenic in children in phase II clinical trials [2].
References 3 & 4 disclose a vaccine against different pathogenic serotypes of serogroup meningococcus based on OMVs which retain a protein complex of 65-kDa. Reference 5 discloses a vaccine comprising OMVs from genetically-engineered meningococcal strains, with the OMVs comprising: at least one Class I outer-membrane protein (OMP) but not comprising a Class 2/3 OMP. Reference 6 discloses OMVs comprising OMPs which have mutations in their surface loops. Reference 7 discloses compositions comprising OMVs supplemented with transferrin binding proteins (e.g. TbpA and TbpB) and/or Cu,Zn-superoxide dismutase. Reference 8 discloses compositions comprising OMVs supplemented by various proteins. References 9 & 10 also describe OMV preparations from meningococcus.
Reference 11 discloses a process for preparing OMV-based vaccines, particularly for serogroup A meningococcus, comprising the following 10 steps: (a) cultivating bacterial cells; (b) concentrating the cultivated cells from step (a); (c) treating the cells with a bile acid salt detergent at a pH sufficiently high not to precipitate the detergent, for inactivating the bacteria, disrupting the outer membrane of the bacteria and forming vesicles of the outer membrane of the bacteria, said vesicles comprising outer membrane components mainly presented in their native form; (d) centrifuging the composition from step (c) at 10,000-20,000×g for about 1 to 2 hours to separate the outer membrane vesicles from the treated cells and cell debris, and collecting the supernatant; (e) performing a high speed centrifugation of the supernatant from step (d) and collecting the outer membrane vesicles in a pellet; (f) re-dispersing the pellet from step (e) in a buffer by stirring at ambient temperature; (g) performing a second high speed centrifugation in accordance with step (e), collecting the outer membrane vesicles in a pellet; (h) re-dispersing the pellet from step (g) in an aqueous medium containing a stabilising agent by stirring at ambient temperature; (i) performing a step-wise sterile filtration through at least two filters of decreasing pore size of the re-dispersed composition from step (h), ending with a filter of pore-size of about 0.2 μm; and (j) optionally including the composition from step (i) in a pharmaceutically acceptable carrier and/or adjuvant composition.
It is an object of the present invention to provide an improved process for preparing OMVs for use in vaccines, in particular a process which can prepare a greater quantity of OMVs in a shorter time, and particularly a process suitable for industrial-scale use.